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Right-side-out vesicles of pig kidney microsomes and amino-acid-sequence-specific antibodies were used to probe the sidedness of the C-terminus and the N-terminus of the catalytic α subunit of Na+/K+-ATPase. Polyclonal antibodies were raised in rabbits against the peptide corresponding to the N-terminal sequence GRDKYEPAAVSE (peptide 1-12) and against peptides corresponding to the C-terminal sequences IFVYDEVRKLIIRRR (peptide 991-1005) and RPGGWVEKETYY (peptide 1005-1016). These antibodies were purified by affinity chromatography on the respective peptide-Sepharose columns. Moreover, antibodies against the N-terminal dodecapeptide GRDKYEPAAVSE were obtained by affinity purification from heteroclonal antibodies against the α subunit of pork kidney Na+/K+-ATPase. These antibodies reacted with native as well as SDS-denaturated Na+/K+-ATPase. When the antibodies were used to probe the sidedness of the sequences in right-side-out vesicles of pig kidney microsomes, the N-terminal peptide 1-12 as well as the C-terminal peptides 991-1005 and 10051016 were found on the cytosolic side. Concanavalin A, however, which interacts with the β subunit, a glycoprotein, reacted with the outside of right-side-out vesicles. [ABSTRACT FROM AUTHOR]

The fluorescing sulfhydryl reagent N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) inactivates purified (Na+ + K+)-ATPase at 20 µM. This inactivation results in a decrease of the ouabain-binding capacity of the enzyme. Treatment of (Na+ + K+)-ATPase, embedded in fight-side-out-oriented vesicles, by DACM does not affect ouabain binding to the enzyme. Incorporation of DACM into the α subunit of (N+ + K+ )-ATPase embedded in right-side-out vesicles is also not affected by the presence or absence of 100 µM ouabain. It is therefore concluded that a sulfhydryl group does not reside within the ouabain-binding site of (Na+ + K+)-ATPase. [ABSTRACT FROM AUTHOR]

The chromium(III) complex of ATP, an MgATP complex analogue, inactivates (Na+ + K+)-ATPase by forming a stable chromo-phosphointermediate. The rate constant k2 of inactivation at 37°C of the β,&gamam;-bidentate of CrATP is enhanced by Na+ (K0.5 = 1.08 mM), imidazole (K0.5 = 15 mM) and Mg2+ (K0.5 = 0.7 mM). These cations did not affect the dissociation constant of the enzyme-chromium-ATP complex. The inactive chromophosphoenzyme is reactivated slowly by high concentrations of Na+ at 37°C. The half-maximal effect on the reactivation was reached at 40 mM NaCl, when the maximally observable reactivation was studied. However, 126 mM NaCl was necessary to see the half-maximal effect on the apparent reactivation velocity constant. K+ ions hindered the reactivation with a K of 70 μM. Formation of the chromophosphoenzyme led to a reduction of the Rb+ binding sites and of the capacity to occlude Rb+. The β,γ-bidentate of chromium(III)ATP (Kd = 8 μM) had a higher affinity than the α,β,γ-tridentate of chromium(III)ATP (Kd = 44 μM) or the cobalt tetramine complex of ATP (Kd = 500 μM). The β,γ-bidentate of the chromium(III) complex of adenosine 5′-[β,γ-methylene]triphosphate also inactivated (Na+ + K+)ATPase. Although CrATP could not support Na+ ,K + exchange in everted vesicles prepared from human red blood cells, it supported the Na+-Na+ and Rb+-Rb+ exchange. It is concluded that CrATP opens up Na+ and K+ channels by forming a relatively stable modified enzyme-CrATP complex. This stable complex is also formed in the presence of the chromium complex of adenosine 5′-[β,γ-methylene]triphosphate. Because the β,γ-bidentate of chromium ATP is recognized better than the α,β,γ-tridentate, it is concluded that the triphosphate site recognizes MgATP with a straight polyphosphate chain and that the Mg2+ resides between the β- and the γ-phosphorus. The enhancement of inactivation by Mg2+ and Na+ may be caused by conformational changes at the triphosphate site. [ABSTRACT FROM AUTHOR]

To characterize pyruvate kinase isoenzymes from cells with the capability to proliferate, this enzyme was purified from yolk and vitelline membrane of unfertilized hen's egg. Pyruvate kinase type M2 from vitelline membrane was obtained in a homogeneous form after a 1150‐fold purification to a specific enzymatic activity of 450 μmol · min−1· mg−1. It was saturated half‐maximally with phosphoenolpyruvate at KPPrv0.5= 0.36 mM phosphoenolpyruvate and was activated by fructose 1,6‐bisphosphate and L‐serine at suboptimal substrate concentrations. After 11000‐fold purification to a specific enzymatic activity of 60 μmol min−1· mg−1, the pyruvate kinase isoenzymes type M2 (KPPrv0.5= 0.32 mM) and M1 (KPPrv0.5= 0.04 mM) were obtained from the yolk substance. Kinetic differences were noted between the pyruvate kinase type‐M2 isoenzymes from vitelline membrane and yolk. A comparison of the amino acid composition of the purified pyruvate kinase isoenzymes from hen's egg revealed that all isoenzymes were related to pyruvate kinase type M1 from chicken breast muscle. The M2‐type isoenzyme from vitelline membrane was related to the M2‐type isoenzyme from chicken tumors, but was not related to the M2‐type pyruvate kinase from chicken lung or liver. Protein kinase C from chicken oviduct phosphorylated in vitro both pyruvate kinase M2 isoenzymes from the unfertilized hen's egg preferably at serine and less at threonine residues. Pyruvate kinase type M1 from egg yolk was a weak substrate of protein kinase C. An activation of pyruvate kinase type M2 from vitelline membrane was observed at suboptimal concentrations of phosphoenolpyruvate under the conditions of phosphorylation, in the presence of phosphatidylserine. [ABSTRACT FROM AUTHOR]

1-Pyreneisothiocyanate was shown to be an inhibitor of Na+/K+-ATPase. Reverse-phase HPLC and activity studies indicated binding of 1-pyreneisothiocyanate at the H4-H5 loop of the α subunit and competition with the fluorescein 5′-isothiocyanate for the E1ATP site. While fluorescein 5′-isothiocyanate, the fluorescent ATP pseudo-analog, was shown to be immobilized at the E1ATP site, there was no possibility to draw any conclusion about the flexibility of the E1ATP site due to its short lifetime. Employing 1pyreneisothiocyanate as a long-lived fluorophore and a label for the E1ATP site, we found that the ATP-binding site of Na+/K+-ATPase and, in fact, the whole large intracellularly exposed H4-H5 loop of the catalytic α subunit is rigid and rotationally immobilized. This has important consequences for the molecular mechanism of the transport function. [ABSTRACT FROM AUTHOR]

To probe the pH value in the microenvironment of the cardiac glycoside-binding site of Na+/K+- ATPase, pH-sensitive fluorescent derivatives of ouabain were synthesized. The fluoresceinyl derivative of ethylenediamino-ouabain CREDO) had a pKs of 6.0 and showed a H--dependent fluorescence change, when its ratio of excitation at 490 nm/450 nm was recorded at 530 nm. Binding of FEDO inactivated Na¯/K+-ATPase at 37°C and pH 7.25 in a slow time-dependent process under the conditions of backdoor phosphorylation with kon of 891 s-1 M-1. The complex dissociated with koff of 0.35×10-3 s-1 resulting in a Kd value of 0.4 μM for the FEDO · enzyme complex. Binding of FEDO was associated with a decrease of the excitatory fluorescence ratio at 490 nm/450 nm which could be used to convert this change into a pH value. A pH value of 5.1 ± 0.2 was calculated to exist in the microenvironment of the FEDO-enzyme complex. This pH value was independent of the pH of the incubation medium used to form the FEDO · enzyme complex. Analysis of the accessibility of the fluorophore in the FEDO · enzyme complex to the dynamic quencher potassium iodide detected a decrease of the Stern­Volmer constant from 6.2 mM-1 (free FEDO) to 1.5 mM-1 (FEDO · enzyme complex} indicating thereby a limited accessibility of the fluorophore to anions. Analysis of the microenvironment of the fluorescein residue of the FEDO · enzyme complex by measurements of the anisotropy and the fluorescence half-life time revealed that both processes differed significantly when H2O was replaced by D:O. We conclude, therefore, that a pH of 5.1 ± 0.2 exists in the vicinity of ouabain that is hidden in the depth of the receptor site when the ouabain, receptor complex has been formed. [ABSTRACT FROM AUTHOR]

Hydrolysis of ATP by Na + /K + -ATPase, a P-Type ATPase, catalyzing active Na + and K + transport through cellular membranes leads transiently to a phosphorylation of its catalytical α -subunit. Surprisingly, three-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp 369 to allow the transfer of ATP's terminal phosphate to its aspartyl-phosphorylation site. In order to get information for how the transfer of the γ -phosphate group of ATP to the Asp 369 is achieved, analogous molecular modeling of the M 4 -M 5 loop of ATPase was performed using the crystal data of Na + /K + -ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr 338 and Ile 760 of the α 2 -subunit of Na + /K + -ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of two ATP binding sites in the open conformation; the first one close to Phe 475 in the N-domain, the other one close to Asp 369 in the P-domain. However, binding of Mg 2+ •ATP to any of these sites in the "open conformation" may not lead to phosphorylation of Asp 369 . Additional conformations of the cytoplasmic loop were found wobbling between "open conformation" <==> "semi-open conformation <==> "closed conformation" in the absence of 2Mg 2+ •ATP. The cytoplasmic loop's conformational change to the "semi-open conformation"-characterized by a hydrogen bond between Arg 543 and Asp 611 -triggers by binding of 2Mg 2+ •ATP to a single ATP site and conversion to the "closed conformation" the phosphorylation of Asp 369 in the P-domain, and hence the start of Na + /K + -activated ATP hydrolysis., Competing Interests: The authors declare there are no competing interests.

Cardiotonic steroids (CTS), long used to treat heart failure, are endogenously produced in mammals. Among them are the hydrophilic cardenolide ouabain and the more hydrophobic cardenolide digoxin, as well as the bufadienolides marinobufagenin and telecinobufagin. The physiological effects of endogenous ouabain on blood pressure and cardiac activity are consistent with the "Na(+)-lag" hypothesis. This hypothesis assumes that, in cardiac and arterial myocytes, a CTS-induced local increase of Na(+) concentration due to inhibition of Na(+)/K(+)-ATPase leads to an increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) via a backward-running Na(+)/Ca(2+) exchanger. The increase in [Ca(2+)](i) then activates muscle contraction. The Na(+)-lag hypothesis may best explain short-term and inotropic actions of CTS. Yet all data on the CTS-induced alteration of gene expression are consistent with another hypothesis, based on the Na(+)/K(+)-ATPase "signalosome," that describes the interaction of cardiac glycosides with the Na(+) pump as machinery activating various signaling pathways via intramembrane and cytosolic protein-protein interactions. These pathways, which may be activated simultaneously or selectively, elevate [Ca(2+)](i), activate Src and the ERK1/2 kinase pathways, and activate phosphoinositide 3-kinase and protein kinase B (Akt), NF-kappaB, and reactive oxygen species. A recent development indicates that new pharmaceuticals with antihypertensive and anticancer activities may be found among CTS and their derivatives: the antihypertensive rostafuroxin suppresses Na(+) resorption and the Src-epidermal growth factor receptor-ERK pathway in kidney tubule cells. It may be the parent compound of a new principle of antihypertensive therapy. Bufalin and oleandrin or the cardenolide analog UNBS-1450 block tumor cell proliferation and induce apoptosis at low concentrations in tumors with constitutive activation of NF-kappaB.

Cardiac glycosides have been used for decades to treat congestive heart failure. The recent identification of cardiotonic steroids such as ouabain, digoxin, marinobufagenin, and telocinobufagin in blood plasma, adrenal glands, and hypothalamus of mammals led to exciting new perspectives in the pathology of heart failure and arterial hypertension. Biosynthesis of ouabain and digoxin occurs in adrenal glands and is under the control of angiotensin II, endothelin, and epinephrine released from cells of the midbrain upon stimulation of brain areas sensing cerebrospinal Na(+) concentration and, apparently, the body's K(+) content. Rapid changes of endogenous ouabain upon physical exercise may favor the economy of the heart by a rise of intracellular Ca(2)(+) levels in cardiac and atrial muscle cells. According to the sodium pump lag hypothesis, this may be accomplished by partial inhibition of the sodium pump and Ca(2+) influx via the Na(+)/Ca(2+) exchanger working in reverse mode or via activation of the Na(+)/K(+)-ATPase signalosome complex, generating intracellular calcium oscillations, reactive oxygen species, and gene activation via nuclear factor-kappaB or extracellular signal-regulated kinases 1 and 2. Elevated concentrations of endogenous ouabain and marinobufagenin in the subnanomolar concentration range were found to stimulate proliferation and differentiation of cardiac and smooth muscle cells. They may have a primary role in the development of cardiac dysfunction and failure because (i) offspring of hypertensive patients evidently inherit elevated plasma concentrations of endogenous ouabain; (ii) such elevated concentrations correlate positively with cardiac dysfunction, hypertrophy, and arterial hypertension; (iii) about 40% of Europeans with uncomplicated essential hypertension show increased concentrations of endogenous ouabain associated with reduced heart rate and cardiac hypertrophy; (iv) in patients with advanced arterial hypertension, circulating levels of endogenous ouabain correlate with BP and total peripheral resistance; (v) among patients with idiopathic dilated cardiomyopathy, high circulating levels of endogenous ouabain and marinobufagenin identify those individuals who are predisposed to progressing more rapidly to heart failure, suggesting that endogenous ouabain (and marinobufagenin) may contribute to toxicity upon digoxin therapy. In contrast to endogenous ouabain, endogenous marinobufagenin may act as a natriuretic substance as well. It shows a higher affinity for the ouabain-insensitive alpha(1) isoform of Na(+)/K(+)-ATPase of rat kidney tubular cells and its levels are increased in volume expansion and pre-eclampsia. Digoxin, which is synthesized in adrenal glands, seems to counteract the hypertensinogenic action of ouabain in rats, as do antibodies against ouabain, for example, (Digibind) and rostafuroxin (PST 2238), a selective ouabain antagonist. It lowers BP in ouabain- and adducin-dependent hypertension in rats and is a promising new class of antihypertensive medication in humans.

The search for an endogenous digitalis has led to the identification of the cardenolides ouabain and digoxin and the bufadienolide marinobufagenin in mammalian tissues and biological fluids. Ouabain's release from adrenal glands is under the control of epinephrine and angiotensin II; hence, its blood concentration changes rapidly on physical exercise. It also is controlled by brain areas sensing cerebrospinal Na+ concentration and apparently the body's K+ content because urinary K+ loss leads to an increase in its plasma concentration as well. Long-term treatment of rats with ouabain results in arterial hypertension, and 50% of Caucasians with low-renin hypertension have increased plasma concentrations of this cardenolide. Levels of digoxin, which is synthesized from acetate in adrenal glands, increase slightly in blood on prolonged exercise. It counteracts the hypertensinogenic action of ouabain in rats, as does the ouabain antagonist PST 2238. The plasma concentration of the bufadienolide marinobufagenin is increased after cardiac infarction. It may show natriuretic properties because it inhibits the alpha1 isoform of Na+/K+-adenosine triphosphatase (ATPase), the main sodium pump isoform of the kidney, much better than other sodium pump isoforms. These effects of endogenous cardiac glycosides are observed at concentrations that do not inhibit the sodium pump. Apparently, Na+/K+-ATPase is used by these steroids as a signal transducer to activate tissue proliferation, heart contractility, arterial hypertension, and natriuresis via various intracellular signaling pathways.

Ouabain, an inhibitor of the sodium pump, has been identified as a constituent of bovine adrenal glands. We were interested whether the release of this cardiotonic steroid is stimulated by physical exercise. Hence, athletes and healthy dogs were subjected to ergometry. Ouabain-like compound (OLC) was measured in venous blood by enzyme-linked immunosorbent assay as well as by (86)Rb+ uptake inhibition (as ouabain equivalents). OLC increased in venous blood of athletes after 15 minutes of ergometry from 2.5+/-0.5 to 86.0+/-27.2 nmol/L (n=51; P<0.001), as did the concentration of a circulating inhibitor of the sodium pump from 7.3+/-1.7 to 129.8+/-51 nmol/L (ouabain equivalents, P<0.05). Half-maximal increase in heart rate and systolic blood pressure occurred at 5.1+/-1.2 nmol/L and at 30+/-1 nmol/L OLC, respectively. On rest, OLC decreased in humans and dogs with a half-life of 3 to 5 minutes. In beagles exposed to moderate exercise on a treadmill for 13 minutes, levels of OLC increased 46-fold (from 3.7+/-0.8 to 166.9+/-91.8 nmol/L; n=6; P<0.005). This effect was suppressed when the dogs had been treated for 3 weeks with the beta1-adrenergic receptor blocker atenolol or the angiotensin-converting enzyme inhibitor benazepril. We conclude that OLC changes rapidly during exercise and is under the control of norepinephrine and angiotensin II.

The structural stability of the large cytoplasmic domain (H(4)-H(5) loop) of mouse alpha(1) subunit of Na(+)/K(+) ATPase (L354-I777), the number and the location of its binding sites for 2'-3'-O-(trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) and p-nitrophenylphosphate (pNPP) were investigated. C- and N-terminal shortening revealed that neither part of the phosphorylation (P)-domain are necessary for TNP-ATP binding. There is no indication of a second ATP site on the P-domain of the isolated loop, even though others reported previously of its existence by TNP-N(3)ADP affinity labeling of the full enzyme. Fluorescein isothiocyanate (FITC)-anisotropy measurements reveal a considerable stability of the nucleotide (N)-domain suggesting that it may not undergo a substantial conformational change upon ATP binding. The FITC modified loop showed only slightly diminished phosphatase activity, most likely due to a pNPP site on the N-domain around N398 whose mutation to D reduced the phosphatase activity by 50%. The amino acids forming this pNPP site (M384, L414, W411, S400, S408) are conserved in the alpha(1-4) isoforms of Na(+)/K(+) ATPase, whereas N398 is only conserved in the vertebrates' alpha(1) subunit. The phosphatase activity of the isolated H(4)-H(5) loop was neither inhibited by ATP, nor affected by mutation of D369, which is phosphorylated in native Na(+)/K(+) ATPase.

Mutation of Arg(423) at the N-domain of Na(+)/K(+)-ATPase resulted in a large decrease of both TNP-ATP and ATP binding. Thus, this residue, localized outside the binding pocket, seems to play a key role in supporting the proper structure and shape of the binding site. In addition, mutation of Glu(472) also caused a large decrease of both TNP-ATP and ATP binding. On the basis of our computer model, we hypothesized that a hydrogen bond between Arg(423) and Glu(472) supports the connection of two opposite halves of the ATP-binding pocket. To verify this hypothesis, we have also prepared the construct containing both these mutations. Binding of neither TNP-ATP nor ATP to this double mutant differed from binding to any of the single mutants. This strongly supported the existence of the hydrogen bond between Arg(423) and Glu(472). Similarly, the conserved residue Pro(489) seems to be substantial for the proper interaction of the third and fourth beta-strands of the N-domain, which both contain residues that take part in ATP binding. Mutation of Asp(443) affected only ATP, but not TNP-ATP, binding, suggesting that these ligands adopt different positions in the nucleotide-binding pocket. On the basis of a recently published crystal structure [Håkansson, K. O. (2003) J. Mol. Biol. 332, 1175-1182], we improved our model and computed the interaction of these two ligands with the N-domain. This model is in good agreement with all previously reported spectroscopic data and revealed that Asp(443) forms a hydrogen bond with the NH(2) group of the adenosine moiety of ATP, but not TNP-ATP.

Endogenous ouabain changes rapidly in humans and dogs upon physical exercise and is under the control of epinephrine and angiotensin II. Hence, the steroid acts as a rapidly acting hormone. A search for a specific binding globulin for cardiac glycosides in bovine plasma resulted in the identification of the d allotype of the micro chain of IgM whose hydrophobic surfaces interact with cardiotonic steroids and cholesterol. Such IgM complexes might be involved in the hepatic elimination of cardiotonic steroids. Thus, differences in the signaling cascade starting at Na(+),K(+)-ATPase must explain any differences in the action of ouabain and digoxin in the genesis of arterial hypertension.

Two glutamate receptor agonists, NMDA (N-methyl-D-aspartic acid) and ACPD (cis-(1S/3R)-1-aminocyclopentane- 1,3-dicarboxylic acid), induce the reactive oxygen species (ROS) production in rat cerebellum granule cells, whereas the third one, 3-HPG (3-hydroxyphenylglycine), decreases this parameter. The simultaneous presence of 3-HPG, together with NMDA or ACPD, prevents the generation of ROS by neuronal cells. A similar effect of these ligands on Na+/K+-ATPase can be demonstrated: NMDA and ACPD inhibited the enzyme activity, but 3-HPG activated Na+/K+-ATPase and prevented its inhibition by NMDA or ACPD. In terms of current classification, NMDA is an agonist of ionotropic glutamate receptors of the so-called NMDA class, whereas ACPD and 3-HPG belong to metabotropic agonists, the former primarily being an activator of metabotropic glutamate receptors (mGluRs) of groups 2 and 3, and the latter, that of mGluRs of groups 1 and 5. Thus, the data presented illustrate the existence of diverse mechanisms of the cross talk between Na+/K+-ATPase and different glutamate receptors, as well as that between glutamate receptors of different classes.

The ATP-binding site of Na(+)/K(+)-ATPase is localized on the large cytoplasmic loop of the alpha-subunit between transmembrane helices H(4) and H(5). Site-directed mutagenesis was performed to identify residues involved in ATP binding. On the basis of our recently developed model of this loop, Ser(445), Glu(446), and Phe(475) were proposed to be close to the binding pocket. Replacement of Phe(475) with Trp and Glu(446) with Gln profoundly reduced the binding of ATP, whereas the substitution of Ser(445) with Ala did not affect ATP binding. Fluorescence measurements of the fluorescent analog TNP-ATP, however, indicated that Ser(445) is close to the binding site, although it does not participate in binding.

The search for endogenous digitalis has led to the isolation of ouabain as well as several additional cardiotonic steroids of the cardenolide and bufadienolide type from blood, adrenals, and hypothalamus. The concentration of endogenous ouabain is elevated in blood upon increased Na(+) uptake, hypoxia, and physical exercise. Changes in blood levels of ouabain upon physical exercise occur rapidly. Adrenal cortical cells in tissue culture release ouabain upon addition of angiotensin II and epinephrine, and it is thought that ouabain is released from adrenal cortex in vivo. Ouabain levels in blood are elevated in 50% of Caucasians with low-renin hypertension. Infusion over several weeks of low concentrations of ouabain, but not of digoxin, induces hypertension in rats. A digoxin-like compound, which has been isolated from human urine and adrenals, as well various other endogenous cardiac glycosides may counterbalance their actions within a regulatory framework of water and salt metabolism. Marinobufagenin, for instance, whose concentration is increased after cardiac infarction, may show natriuretic properties because it inhibits the alpha1 isoform of Na(+)/K(+)-ATPase, the main sodium pump isoform of the kidney, much better than other sodium pump isoforms. In analogy to other steroid hormones, cardiotonic steroid hormones in blood are bound to a specific cardiac glycoside binding globulin. The discovery of ouabain as a new adrenal hormone affecting Na(+) metabolism and the development of the new ouabain antagonist PST 2238 allows for new possibilities for the therapy of hypertension and congestive heart failure. This will lead in turn to a better understanding of the disease on a physiological and endocrinological level and of the action of ouabain on the cellular level as a signal that is transduced to the plasma membrane as well as to the cell nucleus.